Heat stress leads to rapid lipid remodeling and transcriptional adaptations in Nicotiana tabacum pollen tubes

Abstract After reaching the stigma, pollen grains germinate and form a pollen tube that transports the sperm cells to the ovule. Due to selection pressure between pollen tubes, pollen grains likely evolved mechanisms to quickly adapt to temperature changes to sustain elongation at the highest possible rate. We investigated these adaptions in tobacco (Nicotiana tabacum) pollen tubes grown in vitro under 22°C and 37°C by a multi-omics approach including lipidomic, metabolomic, and transcriptomic analysis. Both glycerophospholipids and galactoglycerolipids increased in saturated acyl chains under heat stress (HS), while triacylglycerols (TGs) changed less in respect to desaturation but increased in abundance. Free sterol composition was altered, and sterol ester levels decreased. The levels of sterylglycosides and several sphingolipid classes and species were augmented. Most amino acid levels increased during HS, including the noncodogenic amino acids γ-amino butyrate and pipecolate. Furthermore, the sugars sedoheptulose and sucrose showed higher levels. Also, the transcriptome underwent pronounced changes with 1,570 of 24,013 genes being differentially upregulated and 813 being downregulated. Transcripts coding for heat shock proteins and many transcriptional regulators were most strongly upregulated but also transcripts that have so far not been linked to HS. Transcripts involved in TG synthesis increased, while the modulation of acyl chain desaturation seemed not to be transcriptionally controlled, indicating other means of regulation. In conclusion, we show that tobacco pollen tubes are able to rapidly remodel their lipidome under HS likely by post-transcriptional and/or post-translational regulation.


Introduction
Around 80% of the 4350,000 known plant species are flowering plants (angiosperms) that mainly rely on sexual reproduction to pass on their genes to the next generation (Christenhusz and Byng, 2016). Sexual reproduction is also the prerequisite for seed and fruit set in crop plants. In most angiosperms, the male gametophyte-the pollen containing the sperm cells or the generative cell that later divides into the two sperm cells-double-fertilizes the female gametophyte. To this end, the pollen has to land on the stigma of a pistil and adhere, rehydrate, and germinate there. Since seed plant sperm cells are immotile, they have to be delivered through the female reproductive tissues to the ovule by tip-growing pollen tubes that are formed by the vegetative cells of the pollen (Sprunck, 2020). In some species, pollen tubes have to grow several centimeters to reach the egg-cell containing ovule. Maize (Zea mays) pollen tubes, for example, can grow up to 50 cm in length with a speed of more than 1 cm per hour (Mascarenshas, 1993). Especially for these very long pollen tubes, it is unlikely that enough lipids for the elongation are stored in the pollen grain, but the tubes rather depend on high de novo synthesis for their growth (Ischebeck, 2016). This assumption is supported by the fact that proteins involved in fatty acid synthesis are 5-10 times more abundant in maturing pollen and growing pollen tubes of tobacco (Nicotiana tabacum) than in leaves or roots Ischebeck, 2016).
The process of sexual reproduction in plants is very sensitive to abiotic stresses like heat stress (HS) or cold stress. Male gametophytes are especially sensitive to HS during all stages of their life and are generally more susceptible than female tissues (Hedhly, 2011;Santiago and Sharkey, 2019). One effect of HS on developing pollen is early tapetum degradation likely due to accumulation of reactive oxygen species (ROS) and the consequent disruption of concerted ROS signals (Zhao et al., 2018). Furthermore, anthers can fail to release pollen grains, possibly due to heat-induced changes in cell wall composition, sucrose transport, and water movement (Santiago and Sharkey, 2019). Also, the developing male gametophyte itself can encounter problems at different stages of development (Müller and Rieu, 2016;Raja et al., 2019;Santiago and Sharkey, 2019).
The effects of HS on pollen germination and pollen tube growth are less well studied than those on pollen development but have also been addressed in several studies. It was shown that tomato (Solanum lycopersicum) pollen germination is negatively affected by HS in in vitro experiments (germination and pollen tube growth in medium outside of female tissues); an effect also attributed to elevated ROS levels (Luria et al., 2019). Various sorghum (Sorghum bicolor) genotypes show in vitro pollen germination reductions of 2%-95% under different HSs (Sunoj et al., 2017). In vivo experiments with pollen from rice (Oryza sativa) or pinyon pine (Pinus edulis) gave similar results (Flores-Renter ıa et al., 2018; Shi et al., 2018).
Inhibited pollen tube growth has been observed as well. Experiments on in vivo grown cotton (Gossypium hirsutum) pollen tubes attribute reduced pollen tube growth to HSinduced reduction of soluble carbohydrate content in the pistil (Snider et al., 2011a(Snider et al., , 2011b. Heat-induced pollen tube growth inhibition in rice was described to be likely due to altered auxin homeostasis within the pistil (Zhang et al., 2018). These experiments highlight the importance of biochemical interactions between pollen tubes and the surrounding pistil tissue during stress. However, pollen tube growth inhibition is also observed in vitro. Experiments with tomato pollen showed maximal germination rates at 15 C and maximal pollen tube lengths at 25 C, higher (or lower) temperatures were inhibitory (Karapanos et al., 2010). Another in vitro study in tomato showed that high temperature-induced ROS inhibit pollen tube growth and that elevated flavonol levels can counteract the heatinduced ROS imbalance (Muhlemannet al., 2018). Also, in vitro pollen tube growth of cotton, rice, and Arabidopsis (Arabidopsis thaliana) is inhibited by high temperatures (Boavida and McCormick, 2007;Song et al., 2015;Coast et al., 2016), indicating that growth inhibitions are not solely due to altered crosstalk with the female tissue, but also due to effects in the pollen tube itself.
A challenge all organs of plants have to meet during elevated temperatures is maintaining membrane fluidity and integrity; this holds true for the plasma membrane as well as for intracellular membranes (Niu and Xiang, 2018). Among the challenges, plant membranes have to meet under elevated temperatures are the prevention of bilayer disintegration (because of membrane hyperfluidity under high temperatures) and peroxidation of unsaturated fatty acids by ROS (Higashi and Saito, 2019).
The effects of long-and short-term HS on the leaf lipidome of different species have been reviewed recently (Higashi and Saito, 2019;Lu et al., 2020): levels of glycerophospholipids containing saturated or mono-unsaturated acyl chains increase; while most glycerophospholipid species with polyunsaturated acyl chains decrease. At the same time, triacylglycerol (TG) levels increase.
Nicotiana tabacum used in this study is a versatile model organism that is also very suited to study pollen tube growth. Here, we show that tobacco pollen tubes can grow well at a relatively high temperature of 37 C and monitor the adaptions of the pollen tubes on its lipidome, transcriptome, and metabolome.

Results
Tobacco in vitro pollen tube growth is not inhibited by elevated temperatures to study successful heat adaptation. For this, we analyzed pollen tube elongation under five different conditions ( Figure 1A) with or without HS: 3 h of growth at room temperature (RT; 22 C, 3 h), 6 h of growth at RT (6 h), 3 h growth at RT and then 3 h growth under HS (HS 3 + 3 h), 3 h growth at RT and then 6 h growth under HS (HS 3 + 6 h), and 3 h growth at RT followed by 3 h growth under HS, and finally 3 h at RT for HS relief (HSR; 3 + 3 + 3 h). The chosen time points do not allow a comparison of extended HS and continuous growth at RT, but a comparison between extended HS and HSR can be drawn. Pollen tubes grown at RT for 3 h reached an average length of 0.46 mm and after 6 h they more than doubled their lengths, reaching 1.1 mm (Figure 1, B and C). If shifted to HS after 3 h of RT growth, pollen tube length after 6 h only reached 0.83 mm. This is a growth reduction of around 38% in comparison to RT 6 h, indicating that while pollen tubes can still grow at this temperature their growth is hampered. If subjected to prolonged HS of 6 h, tubes averaged a length of 1.27 mm while HS relieved pollen tubes grew to 1.19 mm.

Relative glycerophospholipid and galactoglycerolipid composition changes under HS
HS usually induces membrane remodeling in order to maintain membrane integrity. Sustaining pollen tube growth under elevated temperatures thus likely requires lipidomic adaptations, too. To assay such a remodeling, pollen tubes were grown in five replicates under the described temperature regimes ( Figure 1A). Lipids were extracted from the tubes and attached medium. Then, glycerolipids (including neutral glycerolipids and galactoglycerolipids), glycerophospholipids, sphingolipids, and sterol lipids (sterol conjugates) were analyzed by ultra-performance liquid chromatography (UPLC)-nanoelectrospray ionization (nanoESI)-tandem mass spectrometry (MS/ MS); free sterols were analyzed by gas chromatography-mass spectrometry (GC-MS). In total, 383 molecular species from 26 lipid subclasses were detected and relative lipid subclassspecific profiles were calculated as percent values of the individual subclasses. Abundances of these subclasses were expressed by normalization of the absolute peak area to the values after 3 h of RT growth (Supplemental Datasets 1-S11). Digalactosylmonoacylglycerol, monogalactosylmonoacylglycerol, sulfoquinovosylmonacylglycerol, sulfoquinovosyldiacylglycerol, phosphatidylinositol (PI)-monophosphate (PIP), PI-bisphosphate (PIP 2 ), and lysophosphatidylserine could not be detected in any of the samples. Ceramide-phosphate (CerP), glucuronosylinositolphosphoceramide (GlcA-IPC), hexosyl-GlcA-IPC, and hexosylhexosyl-GlcA-IPC were also not found.
HS had only mild effects on the relative abundances of most membrane lipid classes ( Figure 2A). For phosphatidic acid (PA), phosphatidylcholine (PC), PI, and phosphatidylethanolamine (PE), only subtle or no changes were observed when the tubes were grown for an additional 3 h at RT or 3 h under HS (total growth of 6 h). After a total growth of 9 h, the relative amounts of PC and PE increased, but their relative accumulation was slightly reduced in heat-stressed tubes when compared with stress-relieved tubes. Stronger relative increases were observed for the other membrane lipids, especially so after prolonged pollen tube growth. Particularly digalactosyldiacylglycerol (DGDG), phosphatidylserine (PS), and TG showed strong relative increases upon heat treatment (Figure 2A). The effects on the synthesis and breakdown of diacylglycerol (DG), an intermediate of several lipid pathways, were less pronounced ( Figure 2A).
Looking at overall saturation levels, the relative proportion of saturated acyl chains increased in all membrane glycerolipid subclasses except for PS and DG, while the relative abundance of polyunsaturated acyl chains decreased ( Figure 2B). Shifting the tubes back to RT partly led to a reversion of the effects in PC, PE, and phosphatidylglycerol (PG) while relative levels in monogalactosyldiacylglycerol (MGDG) and DGDG remained approximately constant.
Regarding the acyl chain composition (Figure 3), a relative increase in most 16:0-and 18:0-containing glycerophospholipid species was observed after 3 h of HS, while species without saturated acyl chains generally decreased in relative abundance. If pollen tubes were shifted back to RT, most species containing 16:0 and 18:0 decreased again; while lipids containing no saturated acyl chains increased.
Monoacylglycerophospholipids that might negatively affect membrane rigidity (Henriksen et al., 2010) were strongly reduced under HS ( Figure 4A). The relative proportion of most monoacylglycerophospholipids containing 16:0 also increased after 3 h of HS (Figure 4, B-F). The relative amounts of 18:2-and 18:3-containing species on the other hand declined leading to a higher saturation ( Figure 4G).
The general trend observed in the galactoglycerolipids MGDG and DGDG is the same as for glycerophospholipids: 16:0-containing lipid species increased, while 18:3-containing lipid species decreased. Stress relief partially reversed the observed effects again for some species but not for all ( Figure 3).
Fewer changes were observed in the acyl chain composition of the glycerolipids DG and TG ( Figure 5A). DG profiles remained more or less constant under all tested conditions. For TG, some changes over time but almost no changes in response to HS were observed.
To further validate the relative increase of TG levels under HS (Figure 2A), and to get information on absolute TG levels, TG content was also quantified by gas chromatographyflame ionization detection (GC-FID) ( Figure 5B). Heatstressed pollen tubes (3 h RT + 3 h HS) produced more than twice the absolute amount of TG as control pollen tubes (6 h RT) did.

Pollen tube sphingolipid abundance increases under HS
Sphingolipids are a highly diverse class of lipids. All sphingolipids consist of a sphingoid base (SPB) that can be subject to different modifications (Michaelson et al., 2016). If the SPB is linked to a 14-30 carbon acyl chain via an amide bond, a ceramide (Cer) is formed. Cers can be linked to a A B C Figure 1 Pollen tubes were subject to different temperature regimes. A, Schematic depiction of the experimental setup. Pollen tubes were grown under five different conditions: either unstressed for 3 or 6 h at RT (22 C; 3 h, 6 h); at RT for 3 h followed by either 3 or 6 h of HS (37 C; 3 + 3 h, 3 + 6 h); or for 3 h at RT, followed by 3 h HS at 37 C and then another 3 h at RT to assay HSR (3 + 3 + 3 h). B, Violin plot of measured pollen tube lengths under the different conditions. Boxplots are shown within the diagram (center line corresponds to median, upper and lower hinge correspond to 25th and 75th percentiles, upper and lower whisker extend to largest/smallest value within 1.5Â inter-quartile range of the respective hinge, and outliers beyond the whiskers are presented individually). ANOVA was performed (significance level a = 0.05), followed by post hoc Tukey's analysis. Results are presented as compact letter display of all pair-wise comparisons. n = 183-201. C, Images of pollen tubes grown under the indicated conditions. Bars = 0.5 mm. head group, consisting either of a hexose (giving rise to the subclass of hexosylceramides; HexCers) or a phosphate attached to an inositol. This inositol can then be further linked to glucuronic acid (GlcA) forming the core structure of glycosylinositolphosphoceramides, which can carry additional sugar residues. Both, the SPBs and the fatty acids can be further modified by double bonds or hydroxyl groups, SPBs and Cers can also be phosphorylated to either SPBphosphates (SPBPs) or Cer-phosphates (Cer-Ps) ( Figure 6A).
Overall, 96 sphingolipid species from 7 lipid subclasses were identified in the samples (Supplemental Datasets S4-S6). The abundance of sphingolipids in tobacco pollen tubes increased over time ( Figure 6B) and HS led to an additional increase of all sphingolipid subclasses. Especially the levels of SPBP (5.8-fold increase when comparing HS 3 + 3 h to RT 6 h and 2.8-fold when comparing extended HS to HSR) and HexCer (30% and 31% increase, respectively) were elevated.
Sphingolipids of Arabidopsis pollen were previously described to be distinct from sporophytic tissues in having HexCer species that predominantly carry a double bond at the C4 position of the SPB instead of a hydroxy group (Luttgeharm et al., 2015). Also, in this study, after 3 h of growth, this double bond was detected in at least 90% of the HexCer species, in 74% of the SPB species and in 15% of the ceramide species ( Figure 6C; for some lipid species, the position of the hydroxyl group could not unambiguously be determined). Interestingly, the occurrence of this modification was slightly reduced under HS. Instead, the relative abundance of species containing a hydroxyl group at this position increased (by 31% when compared with HS 3 + 3 h   to RT 6 h). Another major change observable under HS was the increased hydroxylation at the C2 of the acyl chain of hexosyl-N-acetylhexosaminyl-GlcA-IPCs (Hex-HexNAc-GlcA-IPCs), which was detected in 45% of all Hex-HexNAc-GlcA-IPC species after 6 h growth at RT and was increased to 72% under HS.   diverse cellular processes, including polar cell growth (Simons and Ikonen, 1997;Beck et al., 2007;Simon-Plas et al., 2011;Mamode Cassim et al., 2019). The profile of free sterols in pollen from various species differs greatly from that in leaves or other sporophytic tissues and it was shown that the sterol synthesis pathway in growing tobacco pollen tubes is truncated with only two species being de novo synthesised: presumably methylenepollinastanol and its precursor cycloeucalenol (Villette et al., 2015;Rotsch et al., 2017). Relative abundance of these sterol species increases during pollen tube growth (Rotsch et al., 2017), as can be seen in the presented data ( Figure 7 and Supplemental Datasets S7 and S8). Especially cycloeucalenol accumulated strongly under HS and less at RT. After HSR,  Figure 5 TG accumulates under HS and becomes more saturated. A, The depicted lipid subclasses from pollen tubes grown under different temperature regimes were investigated. Lipid subclass profiles were measured by UPLC-nanoESI-MS/MS. Values are given as mol % of all detected lipid species per subclass. Only major lipid species (41% of total) are depicted. For statistical analysis, ANOVA was performed (significance level a = 0.05), followed by post hoc Tukey's analysis. n = 5. Error bars represent standard deviation. Results are presented as compact letter display of all pair-wise comparisons. B, Absolute quantification of TG from heat stressed and non-stressed pollen tubes per 1 mg of dry pollen. Also shown is the relative contribution of the respective fatty acids to the total TG amount. Measurements were performed with GC-FID and quantified as peak areas. n = 5. Error bars represent standard deviation. ***P 5 0.005; **P 5 0.001; *P 5 0.05; determined by Student's t test.  Sphingolipid composition and modifications are affected by HS. A, Sphingolipids consist of a SPB bound to an acyl chain of variable length (right box) forming a ceramide. They can carry a head group (left box) consisting of a hexose (HexCer), a phosphate (e.g. SPBP), a phosphate fused to inositol (IPC) that can be further linked via GlcA to additional sugars as depicted for a HexNAc-GlcA-IPC that can also be linked to a further hexose (Hex-HexNAc-GlcA-IPC). In addition, sphingolipids can be modified at the SPB by a double bond in the d4 and d8 position or (mutually exclusive with the d4 double bond) a hydroxylation at the C4 carbon atom. Another variable modification that can be detected is a hydroxylation at the C2 of the acyl chain. B, The depicted sphingolipid subclasses from pollen tubes grown under different temperature regimes were investigated. Abundances of the lipid subclasses were measured by UPLC-nanoESI-MS/MS and normalized to their respective values after 3 h RT. C, For each sphingolipid subclass, the percentage-wise occurrence of modifications was determined: HYD base C4, hydroxylation at C4 of the SPB; DB base d4, double bond at the D4 position of the SPB; DB base d8, double bond at the D8 position of the SPB; HYD FA C2, hydroxylation at C4 of the acyl chain; VLCFA, acyl chain derived from a very long chain fatty acid (C 5 20). n = 5. Error bars represent standard deviation. For statistical analysis, ANOVA was performed (significance level a = 0.05), followed by Post-hoc Tukey analysis. Results are presented as compact letter display of all pair-wise comparisons.
increased accumulation ceased and levels remained constant.
The relative levels of sterylglycoside (SG) and acylsterylglycoside (ASG) were also strongly increased by 84% and 42%, respectively, when comparing 3 h of HS to the control treatment. Sterol ester (SE) species on the other hand decreased in their relative levels ( Figure 8 and Supplemental Datasets S10 and S11). When comparing prolonged heat treatment with HSR, HSR caused SE levels to increase again.

Several central metabolites increase under HS
In our study, we also extracted hydrophilic metabolites from pollen tubes grown under the five temperature regimes ( Figure 9A). As pollen tubes cannot be easily washed, some liquid attached to the tubes that could contain secreted metabolites was included in the measurements. We identified 51 metabolites by GC-MS that are mostly part of central metabolism including organic acids, amino acids, and sugars (Supplemental Datasets S12 and S13). In addition, 17 so far unidentified markers were found. Values of all time points were normalized to the values after 3 h of RT. While most metabolites accumulated during prolonged pollen tube growth, not all of these were affected by HS. Sugar levels were only mildly altered with the exception of the sevencarbon sugar sedoheptulose that increased three-fold and sucrose that increased two-fold ( Figure 9B). Among the organic acids, an increase in 2-isopropylmalate, an intermediate in leucine biosynthesis, and 2-oxoglutarate was observed ( Figure 9C). Interestingly, fumarate and malate were not affected by HS but strongly increased after stress relief. Most amino acids increased during HS, including the noncodogenic amino acids b-lactate, c-amino butyrate, and pipecolate ( Figure 9D). Further metabolites that show an increase under HS include, for example glycerol-3-phosphate ( Figure 9E) and PE ( Figure 9F).

HS leads to strong alteration on the transcriptome level
To assess whether the different adaptations are reflected on a transcriptional level, transcriptome analyses of pollen tubes grown for 3 or 6 h at RT or 3 h at RT followed by 3 h at HS were performed by RNA sequencing (Supplemental Dataset S14). The corresponding principal component analysis (PCA) plot is shown in Figure 10A.
Overall, 26,743 genes were detected, 24,013 of which with more than 0.5 counts per million (CPM) in at least 2 libraries and 21,241 of these could be assigned a UniProt protein ID (Table 1). The respective protein sequences were then blasted against the TAIR 10 Arabidopsis protein library, and 19,798 tobacco proteins had Arabidopsis homologs with an Expect value (E-value) 5 10 -5 . The respective Arabidopsis protein identifiers were later used for functional annotation.
In the analysis of pollen tubes grown for 3 versus 6 h at RT, fold-changes (FCs) were calculated. About 561 genes were differentially expressed (defined as log 2 FC 4 1; false discovery rate [FDR] 4 0.005), 306 of which were upregulated and 255 of which were downregulated ( Figure 10B and Supplemental Dataset S15).
In the comparison of heat-stressed versus non-heatstressed pollen tubes, 2,383 genes were differentially expressed, 1,570 of which were upregulated and 813 of which were downregulated ( Figure 10C and Supplemental Dataset S16). For analyses of gene functions, we further explored the second comparison making use of the functional annotation of the Arabidopsis genome and proteome.

GO-term analysis gives first insights
For gene ontology (GO) analysis, the GO terms assigned to the annotated Arabidopsis homologs were analyzed. For the analysis of RT 6 h versus HS 3 + 3 h, 20,370 genes were assigned with 5,994 different GO terms. Reads per kilo base per million mapped reads (RPKM) values of all genes belonging to the same GO term were summed up and averaged for the respective condition to calculate log 2 FCs.
A selection of the most changed GO terms between RT 6 h and HS 3 + 3 h is presented in Table 2, grouped by subontology (for complete lists, see Supplemental Datasets S17-S20). Among them are several heat shock-related GO terms containing many upregulated genes and only few downregulated genes. The terms include "functions in Hsp90 protein binding," "has unfolded protein binding," and "involved in response to heat." Another strongly changed GO term is "involved in response to ROS." Other changed GO terms like "has pre-mRNA 3'-splice site binding" and "involved in positive regulation of mRNA splicing, via spliceosome" might hint at a role of alternative splicing under HS, which has been shown before, for example in tomato pollen (Keller et al., 2017). Interestingly, several changed GO terms suggest that auxin metabolism and/or signaling might be affected ("has auxin receptor activity," "involved in auxin catabolic process," and "involved in regulation of auxin biosynthetic process").

Transcripts of transcriptional regulators are strongly affected
The GO term analysis revealed the term "has transcription coactivator activity" to be 3.2-fold upregulated. The terms "functions in transcription regulatory region DNA binding" and "has DNA-binding transcription factor activity" showed 34 of 395 and 72 of 654 differentially upregulated, but also 31 and 15 downregulated genes, respectively. The total transcript abundance within these terms was, however, only   Table 3 and Supplemental Dataset S22). The highest proportion of upregulated genes was found among the heat shock TFs (HSF, 9 out of 19) and DNA-binding protein phosphatases (DBP, 6 out of 10). Furthermore, the TF families, MYB-related and AP2-EREBP, showed a comparably high number of upregulated genes (14 and 9, respectively).

Transcripts related to lipid metabolism are barely affected by HS
To find out whether lipid adaptations are mirrored on a transcriptional level, a list of 746 Arabidopsis genes with a role or putative role in glycerolipid, glycerophospholipid, sphingolipid, and sterol lipid metabolism was compiled from several sources (see "Materials and methods" and Supplemental Dataset S23). Comparing our transcript data to this list, 713 tobacco transcripts were assigned a putative function in lipid metabolism (Supplemental Dataset S24). Of these, only 43 genes were differentially expressed, 20 of which were upregulated, and 23 downregulated (Table 4). This result already indicates that lipid adaptations are not strongly reflected on a transcriptional level. To take a closer look at the differentially expressed genes (DEGs), lipid genes were grouped according to their pathways.

Fatty acid synthesis is not transcriptionally upregulated
Pollen tube growth requires fatty acid synthesis in the plastids to cope with the increasing need for membrane lipids in growing pollen tubes (Ischebeck, 2016). Under HS, pollen tubes seem to need slightly more membrane lipids, as several minor lipid classes, as well as TG, levels accumulated more at high temperature (Figures 2A and 5B). However, neither of the pathways leading to acetyl-CoA synthesis via the pyruvate dehydrogenase nor pyruvate decarboxylase seems to be strongly affected (Supplemental Dataset S24). The same is true for fatty acid synthesis itself, as only one relevant gene, one of six putative ketoacyl-ACP reductases (KAR) was upregulated. It was previously shown that downregulation of the ketoacylsynthase KASII/FAB1, which is responsible for the elongation from C16 to C18 acyl chains, leads to strongly increased levels of C16 acyl chains in Arabidopsis (Pidkowich et al., 2007). Interestingly, none of the four isoforms was considerably downregulated in tobacco pollen tubes under HS, despite the increase of 16:0 acyl chains across lipid subclasses (Figure 3).
What is more, no differentially expressed fatty acid desaturases (FADs) were detected in our screen. FAD2 and FAD3, the desaturases that catalyze in PC the desaturation from 18:1 to 18:2 and from 18:2 to 18:3, respectively, are Red lines indicate threshold for differential expression (abs. log 2 FC 4 1, outside of vertical lines and FDR 5 0.005, above horizontal line). n = 3. Number of detected genes in total and in the two comparisons and number of total detected genes (unfiltered). For DE analyses, genes were filtered for a CPM 5 0.5 in at least two of the respective analyzed libraries. Genes were annotated a UniProt identifier (UniProt ID), some genes could not be annotated, number of annotated genes is given. Annotated Tobacco proteins were blasted against Arabidopsis to find homologs, only hits with E-value 5 10 -5 were considered. Number of genes with an Arabidopsis protein homolog hit is given.
not downregulated but several isoforms were slightly upregulated.

The transcriptome does not reflect adaptations in glycerolipid and glycerophospholipid profiles
The expression levels of enzymes involved in glycerolipid metabolism gave contrasting results. While some individual isoforms were differentially expressed (Table 4), only few clear trends could be observed. Two putative PA phosphatases homologous to Arabidopsis LPP1/PAP1 had very high abundance levels and increased by 25% and 55% during HS, respectively. Also two of three CGI58-type lysophosphatidic acid (LPA) acyltransferases that are involved in membrane and neutral lipid homeostasis (Ghosh et al., 2009;James et al., 2010) were slightly upregulated by 32% and 35%, respectively (Supplemental Dataset S19). Likewise, no clear trends for genes involved in galactoglycerolipid metabolism were observed. Transcripts important for sulfolipid metabolism were not detected, indicating again that these lipids do not occur in tobacco pollen tubes. A selection of GO terms grouped by sub-ontologies (molecular function, cellular component, and biological process) that show a strong log 2 FC between pollen tubes grown for 6 h at 22 C and tubes grown for 3 h at 22 C followed by 3 h at 37 C. GO terms were quantified summing RPKM values of all detected genes within one GO term (number of genes is given) for the respective condition (6 h 22 C or 3 h 22 C + 3 h 37 C) and log 2 FC of the summed RPKMs was calculated. P-values were calculated using Student's t test on summed RPKM values of the two conditions. Number of DEGs (log 2 FC 4 j1j, FDR 5 0.005 according to DE analysis with EdgeR, Supplemental Datasets S12-S15) within the GO term are given.

Transcripts hint at a dynamic turnover of TG upon HS
Taking a closer look at TG metabolism, we found that DG acyltransferase 1 (DGAT1), DGAT2, and DGAT3 homologs are strongly transcriptionally upregulated after 3 h of HS. However, a homolog of the major TG lipase SUGAR DEPENDENT 1 (SDP1) is also upregulated, so is another putative TG lipase. Upregulated genes also include some known LD-localized proteins; a putative 11-b-hydroxysteroid dehydrogenase (HSD) is very strongly upregulated (125-fold), which is interesting as previously no HSDs were found on LDs of tobacco pollen tubes on the protein level (Kretzschmar et al., 2018). Also, an oleosin, a lipid dropletassociated protein (LDAP) and a putative lipid dropletassociated hydrolase (LDAH) (Kretzschmar et al., 2020) show upregulation (Table 4); while another putative oleosin family protein is downregulated. Some low but significant changes can be observed in the scaffold protein plant UBX-domain-  List of all differentially expressed tobacco genes with a putative involvement in lipid metabolism. Given are the Tobacco gene name, the annotated UniProt ID Tobacco protein name, the Arabidopsis homolog with the highest sequence similarity (gene name and protein name), and the assigned putative Tobacco protein function according to the Arabidopsis homology. Differential expression analysis was performed with EdgeR, log 2 FCs, and FDRs are given.
Several genes involved in sterol and sphingolipid metabolism show differential expression A transcript encoding a methylsterol monooxygenase 2 (SMO2)-like isoform showed eight-fold upregulation. SMO proteins are involved in sterol synthesis, to be more precise SMO2-1 and SMO2-2 are involved in the reaction from 24methylenelophenol to episterol (precursor of campesterol, brassinosteroids, and brassicasterol) and from 24-ehtylenelophenol to d7-avenasterol (that can be converted to isofucosterol, sitosterol, and stigmasterol). Furthermore, a putative d(24)-sterol reductase shows two-fold upregulation.
Regarding sphingolipid metabolism, a D8-fatty-acid desaturase-like protein that shares 62% sequence identity with Arabidopsis SPHINGOID LONG CHAIN BASE DESATURASE 2 (SLD2) was two-fold downregulated, while other homologs of this enzyme showed upregulation (Table 4 and Supplemental Dataset S24). The transcript of a putative SPB hydroxylase 2 (SBH2) that inserts a hydroxyl group at the C4 position showed two-fold upregulation. One of three isoforms of dihydrosphingosine D-4 desaturases catalyzing a competing reaction, the insertion of a double bond at the D4 position, was downregulated by 31%, while the other isoforms displayed little change.

DEGs suggest involvement of hormones in HS adaptation
Some other interesting DEGs that have not been covered so far are highlighted in Table 5. The gene displaying the strongest upregulation is annotated as "uncharacterized protein" in tobacco and its closest Arabidopsis homolog, AT3G10020, is annotated as "plant/protein." Publications suggest that it is a stress-responsive gene. Strongest downregulation was observed for a homolog of BON ASSOCIATION PROTEIN 2 (BAP2), a reported inhibitor of programmed cell death (Yang et al., 2007).
While auxins were already mentioned in the GO terms, some other hormones might also play a role in HS adaptation of tobacco pollen tubes. Strong upregulation was observed for a homolog of Arabidopsis JASMONATE-INDUCED OXYGENASE2 (JAO2), annotated as a protein with similarity to flavonol synthases and involved in the detoxification of polycyclic aromatic hydrocarbons (Hernández-Vega et al., 2017). JASMONATE-ZIM-DOMAIN PROTEIN 8 (JAZ8) on the other hand was transcriptionally downregulated. The transcript of a putative homolog of BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), a receptor-like kinase in brassinosteroid sensing, was downregulated under HS.

Discussion
The tobacco pollen tube transcriptome shows expected but also unique adaptations to HS As shown in several previous studies on plants, HS leads to a rapid and strong remodeling of the transcriptome (Kotak et al., 2007;Mittal et al., 2012;Rahmati Ishka et al., 2018). One reaction conserved across prokaryotes and eukaryotes is the upregulation of heat shock proteins (HSPs) (Jacob et al., 2017). Accordingly, most transcripts strongly upregulated in response to HS encode HSPs in this study, indicating that the applied temperature regime imposes HS to the pollen tubes (Supplemental Dataset S16). This upregulation has also been found to be a key response under HS in developing pollen, prior to pollen tube growth (Fragkostefanakis et al., 2016;Keller et al., 2018).
However, we also found differences between HS responses in tobacco pollen tubes and vegetative tissues of Arabidopsis, as several homologs of heat-induced genes in Arabidopsis are expressed, but not upregulated upon HS in tobacco pollen tubes. These include genes encoding for LATE EMBRYOGENESIS ABUNDANT proteins, which protect other proteins and membranes especially during desiccation but are also upregulated by HS (Priya et al., 2019). On the contrary, we found genes upregulated that have so far not been associated with HS. These include, for example a flotillin-like gene homologous to Arabidopsis FLOT2 (Table 5). Flotillins are involved in formation of membrane microdomains (Haney and Long, 2010;Li et al., 2012). These data suggest that flotillins might also play an important role during pollen tube growth, maybe especially so under stress. The presence of sterol-rich membrane microdomains containing flotillin-like protein in rice pollen has recently been shown (Han et al., 2018).

Heat-induced glycerolipid remodeling does not appear to be strongly transcriptionally controlled
The expression data shows no conclusive picture as to how the membrane lipid composition is altered. Nevertheless, several conclusions can be drawn from membrane lipid remodeling: Especially striking is the increase of the saturated acyl chains 16:0 and 18:0, while the ratio of monounsaturated to polyunsaturated acyl chains is not severely altered. This result suggests a regulation of fatty acid synthesis in the plastids, where it is determined if the acyl carrier protein (ACP)-connected acyl chains are initially elongated and desaturated. Only after elongation and desaturation from 16:0 to 18:1, the acyl chains can be precursors for further extraplastidial desaturation.
Arabidopsis and likely also other plant species harbor three types of KAS with only one, KASII/FAB1, being responsible for the elongation of 16:0-ACP to 18:0-ACP (Wu et al., 1994;Pidkowich et al., 2007). KASII/FAB1 would thus pose a good target for regulation. Regulation of the D9 desaturase FAB2 could be a further factor, as it introduces the first double bond to form 18:1. Furthermore, thioesterases might play a role, as knockout of FATB leads to a strong reduction of saturated acyl chains (Bonaventure et al., 2003) and knockdown of the two FATA genes also influences acyl composition (Moreno-P erez et al., 2012).
One can speculate that during HS the de novo synthesised fatty acids are even stronger saturated than reflected by the membrane lipid composition, as some acyl chains were likely already synthesised prior to HS. This would imply a rapid and strong adaptation of the above-mentioned enzymes, likely through protein degradation, posttranslational modification, or a direct influence of temperature on the enzymatic activities. A transcriptional regulation is, however, unlikely, as respective transcripts were little or not changed (Supplemental Dataset S24).

Role of PS and galactoglycerolipids in HS adaptation
Some lipid subclasses increased comparably stronger under HS. This is especially true for PS and DGDG. PS is found in the cytosolic leaflets of the plasma membrane and in Selection of differentially expressed Tobacco genes (including the gene with the strongest upregulation and downregulation, respectively), the annotated UniProt ID Tobacco protein name, the Arabidopsis homolog with the highest sequence similarity (gene name and protein name), and the assigned putative Tobacco protein function according to the Arabidopsis homology. Differential expression analysis was performed with EdgeR, log2FC, log 2 CPM, and FDRs are given. endosomes and is negatively charged. It can form nanodomains in the plasma membrane that are important for Rho signaling (Platre et al., 2019). These small G proteins have also been shown to be important for the regulation of pollen tube growth , but it remains to be studied if they also depend on PS, how PS is distributed in the pollen tube, and if this distribution changes under HS. Galactoglycerolipids are the most abundant lipids of thylakoids and there is strong galactoglycerolipid remodeling under temperature stress in Arabidopsis (Chen et al., 2006;Higashi et al., 2015) and tomato leaves (Spicher et al., 2016). In agreement with studies concerning other classes of membranes lipids, the level of unsaturation in galactoglycerolipids is inversely associated with growth temperature. Furthermore, heat acclimation at 38 C in wild-type Arabidopsis increases DGDG, the DGDG-to-MGDG ratio, and the saturation level of DGDG (Higashi et al., 2015). Also, a role for galactoglycerolipids in acquired thermotolerance was already described in a study in 2006, when the authors did a mutant screen for plants defective in the acquisition of thermotolerance and found a mutant of DGD1 (Chen et al., 2006). While pollen tubes contain plastids, these harbor no thylakoids (Staff et al., 1989) and galactoglycerolipids in pollen tubes were discussed to be especially important in extraplastidial membranes, where they are also formed in phosphate-starved vegetative tissues (Härtel et al., 2000). Evidence for the abundance of galactoglycerolipids in the male gametophyte comes from glycerolipid profiling of lily (Lilium longiflorum) pollen tubes before and after elongation revealing a 5.7-fold increase in DGDG and a 2.8-fold increase in the MGDG levels (Nakamura et al., 2009). The use of the MGDG synthase inhibitor galvestine-1 further highlighted an important role of galactoglycerolipids in pollen tube growth and the use of a specific antibody indicated a DGDG localization at the periphery of Arabidopsis pollen tubes most probably at the plasma membrane (Bott e et al., 2011). In addition, the fact that galactoglycerolipids account for 11% of all membrane-forming glycerolipids in tobacco pollen tubes  speaks for their presence in extraplastidial membranes.

Transcriptome and lipidome suggest dynamic adaptations in LD-turnover and TG metabolism
While transcripts involved in lipid metabolism were mostly unaffected by HS, transcripts coding for genes involved in TG turnover and LD biology showed more dynamic adaptations to HS (Supplemental Dataset S24). An increase of TG levels under HS has already been observed, for example, in the algal species Nannochloropsis oculata (Converti et al., 2009), Ettlia oleoabundans (Yang et al., 2013), Coccomyxa subellipsoidea C169 (Allen et al., 2018), or Chlamydomonas reinhardtii (L egeret et al., 2016), but also in Arabidopsis leaves (Higashi et al., 2015;Shiva et al., 2020) and seedlings (Mueller et al., 2015), and tomato fruits (Almeida et al., 2021). In Arabidopsis seedlings, PHOSPHOLIPID:DIACYLGLY CEROL ACYLTRANSFERASE1 (PDAT1), an enzyme transferring a fatty acid from PC to DG to yield TG, is necessary for heat-induced TG accumulation. Also, the pdat1 mutant seedlings were more sensitive to HS, indicating that PDAT1-mediated TG accumulation mediates thermotolerance (Mueller et al., 2017). Another interesting fact hinting at an involvement of not just TG but LDs as organelles in HS adaptations is the 125-fold upregulation of a putative HSD5 homolog (Table 4) suggesting an involvement of LDs in HS adaptation beyond TG accumulation. HSDs, also called steroleosins, are important for plant development and are involved in stress responses as well as wax metabolism Zhang et al., 2016;Shao et al., 2019). Steroleosins are presumably involved in brassinosteroid metabolism  but were previously not found on LDs of tobacco pollen tubes (Kretzschmar et al., 2018). In this study, only two transcripts with very low expression levels were detected in nonstressed conditions.

Changes in sterol lipid and sphingolipid metabolism might contribute to heat adaption
In contrast to TG that increases during HS and might therefore act as a sink for acyl chains, SE species are decreased, indicating that free sterols are released during heat adaptation and could help to adapt membrane fluidity (Dufourc, 2008). Free sterols could furthermore be converted to SG and ASG species, which were shown to increase upon HS. So far, no plant sterol esterase has been described  and it is not clear if SE breakdown is regulated transcriptionally.
Alterations in sphingolipid composition on the other hand could at least in part be explained by changes in transcript levels-the slight increase of hydroxylation versus desaturation at the C4 position of the SPB in HexCers is in line with the respective changes in the transcripts coding for the responsible enzymes (Table 4 and Supplemental Dataset S24). The increased hydroxylation of the SPB moiety in HexCers and the acyl chain in Hex-HexNAc-GlcA-IPCs could stabilize sphingolipid-containing microdomains through their ability to form lateral hydrogen bonds with other sphingolipids or free sterols (Mamode Cassim et al., 2019).
The strong increase of SPBP on the other hand is not reflected on the transcript level as none of the most abundant SPB kinases are strongly upregulated (Supplemental Dataset S24). The production of SPBP is thought to play a role in balancing the levels of putatively toxic SPBs. Furthermore, SPB breakdown requires its phosphorylation to SPBP followed by degradation to PE and palmitic aldehyde by SPBP lyase. Furthermore, SPBP could have signaling function (Puli et al., 2016).

Metabolomic adaptations might facilitate thermotolerance
In previous studies on heat-stressed shoot tissues of Arabidopsis (Kaplan et al., 2004;Harsh et al., 2016;Zinta et al., 2018;Lawas et al., 2019), it was found that different sugars accumulated, including glucose, fructose, and in some cases sucrose. In tobacco pollen tubes, the heat-induced accumulation of sugars was rather modest in comparison and the somewhat higher increase in sucrose has to be considered with care, as sucrose was also contained in the growth medium ( Figure 9). Interestingly though, we found a strong increase in sedoheptulose, a seven carbon sugar normally occurring in its phosphorylated form in the Calvin-Benson cycle and the pentose phosphate pathway. It is unclear if this sugar has any protective function, but it was also found to highly accumulate in the alga Phaeodactylum tricornutum under nitrogen deprivation (Popko et al., 2016).
In addition, an increase of several free amino acids was observed under HS (Figure 9). Proline levels, however, stayed relatively stable and although it accumulates very strongly after 6 h of pollen tube growth, it does so under normal as well as stress conditions. This is interesting, as proline was found to be clearly heat-inducible in studies on shoot tissues (Harsh et al., 2016;Zinta et al., 2018;Lawas et al., 2019). However, a study on potato (Solanum tuberosum) leaves showed that HS alone does not have substantial influence on proline levels, neither in stress-susceptible nor in tolerant potato cultivars. In the study, proline only accumulated after drought or combined HS and drought stress (Demirel et al., 2020). The same study observed an increase in lysine in one of the stress-susceptible cultivars following HS. It is possible that pipecolate, similar in structure to proline, plays a role in adaptation to heat in pollen tubes, as pipecolate was more heat-responsive than proline in this study. Pipecolate was also shown to strongly accumulate during tobacco pollen development (Rotsch et al., 2017) and it increases in shoot tissues upon pathogen infection (Návarová et al., 2013;Ding et al., 2016).

Conclusion
Adaptation of tobacco pollen tubes to HS appears to be a complex and multi-layered trait that requires intricate interplay of different cellular processes. A rapid lipid remodeling that is not controlled transcriptionally is equally as important as metabolomic adaptations and transcriptional changes. Therefore, this study with several large datasets is a useful resource for future studies on HS tolerance.

Plant material and growth conditions
Tobacco (N. tabacum L. cv. Samsun-NN) plants were grown in the greenhouse as previously described (Rotsch et al., 2017): plants were kept under 14 h of light from mercuryvapor lamps in addition to sunlight with light intensities of 150-300 mmol m -2 s -1 at flowers and 50-100 mmol m -2 s -1 at mid-height leaves. Temperature was set to 16 C at night and 21 C during the day with a relative humidity of 57%-68%.
Pollen was first pooled for each experiment and was derived from 5 to 20 plants and 50 to 200 flowers. The pooled pollen was weighed and rehydrated for 10 min in liquid pollen tube medium (5% [w/v] sucrose, 12.5% [w/v] PEG-4000, 15 mM MES-KOH pH 5.9, 1 mM CaCl 2 ,1 mM KCl, 0.8 mM MgSO 4 , 0.01% [w/v] H 3 BO 3 , 30 mM CuSO 4 ) modified from (Read et al., 1993). Aliquots of the suspension containing a defined amount of pollen (indicated below for each experiment) were spread onto cellophane foil (Max Bringmann KG, Germany) and placed on 50 mL of solid pollen tube medium (2% [w/v] Agarose, 5% [w/v] sucrose, 6% [w/v] PEG-4000, 15 mM MES-KOH pH 5.9, 1 mM CaCl 2 ,1 mM KCl, 0.8 mM MgSO 4 , 0.01% [w/v] H 3 BO 3 , 30 mM CuSO 4 ) inside square petri dishes (120 Â 12017 mm with vents, Greiner Bio-One, Kremsmünster, Austria), sealed with MicroporeTM (3 M; Saint Paul, MN, USA). All pollen tubes were grown for 3 h at RT. Control pollen tubes were kept at RT for another 3 h, while heat stressed-pollen tubes were transferred to 37 C for 3 h. For HSR, pollen tubes were then transferred to RT again for the next 3 h, while nonrelieved pollen tubes remained at 37 C. Pollen tubes were harvested using a spatula and forceps, and directly transferred into extraction solution for lipid or metabolite analysis. For RNA extraction, the material was immediately flash-frozen for further processing.
(1) Correction of the absolute peak area with the naturally occurring proportion of the 13 C isotopes (isotopic correction factor, icf); (2) Addition of icf-corrected absolute peak area from corresponding mass transitions; (3) Total peak area of a lipid subclass (addition of all detected molecular species of the respective lipid subclass) and normalization to the values after 3 h of RT growth (relative abundance compared with RT 3 h); relative proportion of the lipid species-specific peak area on the total peak area of the respective lipid subclass (relative lipid subclass profile).

Lipid extraction and fatty acid methyl esterification
For analyses by GC-FID, total lipids from pollen tubes grown from 20 mg of dry pollen were extracted by methyl-tert-butyl ether (MTBE) extraction (modified from Matyash et al., 2008). Pollen tubes were harvested into 2 mL of preheated propan-2-ol (75 C) and kept at 75 C for 5-10 min. About 0.05 mg triheptadecanoate (Merck KGaA, Darmstadt, Germany) in 50 mL chloroform as the internal standards for absolute quantification was directly added to the propan-2ol. Propan-2-ol was transferred to a new vial and evaporated under N 2 stream and the pollen tubes were covered with 2 mL of 3:1 (v/v) MTBE/methanol. Pollen tube tissue was disrupted with a spatula, vortexed, and then shaken at 4 C for 1 h. After 5 min of centrifugation at 1,000 Â g, the supernatant was transferred to the vial with the evaporated propan-2-ol and 1 mL of 0.9% (w/v) NaOH was added. Samples were vortexed and centrifuged again before the upper phase was transferred to a new vial. Solvent was evaporated under a N 2 stream and samples were dissolved in 250 mL of 65:56:8 (v/v/v) chloroform/methanol/water. The total lipids were then separated by thin layer chromatography (TLC). About 80 mL of the dissolved samples were spotted with a TLC spotter to TLC plates (TLC Silica gel 60, Merck KGaA). For extraction of TG, plates were run in 80:20:1 (v/v/v) hexane/diethylether/acetic acid. The bands comigrating with the TG standard were scratched out. To obtain fatty acid methyl esters (FAMEs), samples were then subjected to 1 mL FAME reagent (2.5% [v/v] H 2 SO 4 , 2% [v/ v] dimethoxypropane in 2:1 [v/v] methanol/toluene) and under constant shaking incubated at 80 C in a water bath for 1 h. The reaction was stopped by adding 1 mL of saturated NaCl solution and vortexing. FAMEs were then extracted by adding 1 mL of hexane, centrifuging 10 min at 2,000 Â g, and transferring the upper phase to a new vial. Hexane was evaporated and samples were resuspended in 25 mL of acetonitrile for subsequent GC-FID analysis.

Central metabolite and sterol extraction and derivatization
Primary metabolite and sterol extraction were performed as previously described (Rotsch et al., 2017). Per time point, pollen tubes grown from 5 mg of pollen were harvested and freeze-dried overnight. After tissue disruption, 500 mL extraction solution (32.25:12.5:6.25 [v/v/v] methanol/chloroform/ water) was added per sample, vortexed, and incubated for 30 min at 4 C under constant shaking. Supernatant was transferred to a new tube and pollen tubes were extracted again with another 500 mL of extraction solution. Incubation was repeated and supernatants were combined. About 0.0125 mg allo-inositol in 0.5 mL water was added, incubated again at 4 C for 30 min, centrifuged 5 min at full speed, and the aqueous phase containing the metabolites was transferred. About 20 mL was evaporated under a N 2 stream and used for derivatization with 15 mL of methoxyamine hydrochloride in pyridine (30 mg/mL) overnight at RT. Derivatization with 30 mL N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) followed for at least 1 h to obtain methoxyimino and trimethylsilyl derivatives of the metabolites (Bellaire et al., 2014).
For the analysis of sterols, 2 mL of 3:1 v/v MTBE/methanol and 1 mL of 0.9% (w/v) NaCl were added to 200 mL of the organic phase and evaporated under a N 2 stream. Samples were dissolved in 20 mL pyridine, 10 mL of which were used for MSTFA derivatization with 10 mL of MSTFA 1-6 h prior to analysis.

GC-FID and GC-MS
GC-FID analysis of FAMEs was performed as described (Hornung et al., 2002): an Agilent GC 6890 system (Agilent, Waldbronn, Germany) coupled with an FID detector equipped with a capillary DB-23 column (30 m Â 0.25 mm, 0.25 mm coating thickness, Agilent) was used. Helium served as the carrier gas (1 mL/min), with an injector temperature of 220 C. The temperature gradient was 150 C for 1 min, 150 C-200 C at 15 K min -1 , 200 C-250 C at 2 K min -1 , and 250 C for 10 min.
For quantification, peak integrals were determined using Agilent ChemStation for LC 3D systems (Rev. B.04.03) and used to calculate absolute amounts of TG as well as relative fatty acid contributions.
For the measurement of central metabolites and sterols, GC-MS measurements were performed as previously described in Touraine et al. (2019) for metabolites and Berghoff et al. (2021) for sterols. If the metabolites were not identified by an external standard, the spectra were identified with the Golm metabolome database (GMD) and the National Institute of Standards and Technology spectral library 2.0f. The chemical information on metabolites identified with the GMD can be obtained at http://gmd.mpimpgolm.mpg.de/search.aspx (Kopka et al., 2005). Due to the high levels, sucrose was measured in separate runs using only one-tenth of the sample as used for the regular runs.

RNA extraction, library preparation, and sequencing
Total RNA was extracted from 200 mg of pollen tubes per sample using Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA). RNA-seq libraries were performed using the non-stranded mRNA Kit from Illumina (San Diego, CA, USA; Cat. No. RS-122-2001). Quality and integrity of RNA were assessed with the Fragment Analyzer from Advanced Analytical (Heidelberg, Germany) by using the standard sensitivity RNA Analysis Kit (Agilent, Santa Clara, CA, USA; DNF-471). All samples selected for sequencing exhibited an RNA integrity number 48. After library generation, for accurate quantitation of cDNA libraries, the fluorometricbased system QuantiFluordsDNA (Promega, Madison, WI, USA) was used. The size of final cDNA libraries was determined using the dsDNA 905 Reagent Kit (Fragment Analyzer, Advanced Bioanalytical) exhibiting a sizing of 300 bp on average. Libraries were pooled and sequenced on an Illumina HiSeq 4000 (Illumina), generating 50 bp singleend reads (30-40 Mio reads/sample). The data can be found under the GEO ID: GSE153474 and the link https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE153474.

Differential gene expression analysis
Read counts were analyzed in the R/Bioconductor environment (Release 3.10, www.bioconductor.org) using edgeR package (version 3.28.1) (Robinson et al., 2009;McCarthy et al., 2012) gene names were translated to UniProt protein identifiers. For more detailed functional analyses, these tobacco proteins were blasted against the TAIR 10 Arabidopsis protein library (TAIR10, https://www.arabidopsis.org/down load_files/Proteins/TAIR10_protein_lists/TAIR10_pep_20101 214) using Protein-Protein BLAST 2.5.0 + with a maximum target sequence of 1. Only those hits with an Expect value (E-value describes the amount of hits to be expected by chance for the respective database size) 510 -5 were considered. The obtained Arabidopsis AGI-codes were then assigned GO-terms for GO-term analysis. The GO term annotations were obtained from the Arabidopsis Information Resource (www.arabidopsis.org) in a version updated on 1 January 2020.
To analyze genes with a putative involvement in lipid metabolism, a compiled list of lipid genes was generated using The Arabidopsis Acyl-Lipid Metabolism Website , KEGG pathway (https://www.genome.jp/kegg/ pathway.html, latest update 10 March 2020), and genes from Kretzschmar et al. (2020), Kelly and Feussner (2016), and Luttgeharm et al. (2016).

Statistical analyses
Statistical analyses were performed as indicated for the respective experiments. For multiple comparisons, analysis of variance (ANOVA) was performed, followed by post hoc Tukey analysis. Results are presented as compact letter display of all pair-wise comparisons. Unpaired two-sample t tests were performed if just two means were compared. Results are presented as * (P 5 0.05), **(P 5 0.01), and ***(P 5 0.005).

Supplemental data
The following materials are available in the online version of this article.
Supplemental Dataset S2. Glycerolipids: icf-corrected peak areas (partially summed) on the molecular species level and the lipid subclass level.
Supplemental Dataset S3. Glycerolipids: relative peak areas as % of all icf-corrected peak areas of the respective lipid subclass.
Supplemental Dataset S5. Sphingolipids: icf-corrected peak areas (partially summed) on the molecular species level and the lipid subclass level.
Supplemental Dataset S6. Sphingolipids: relative peak areas as % of all icf-corrected peak areas of the respective lipid subclass.
Supplemental Dataset S10. Sterol conjugates: icfcorrected peak areas on the molecular species level and the lipid subclass level.
Supplemental Dataset S11. Sterol conjugates: relative peak areas as % of all icf-corrected peak areas of the respective lipid subclass.
Supplemental Dataset S21. List of Arabidopsis transcription regulators.
Supplemental Dataset S22. Transcriptomics: list of all detected putative transcription regulators.
Supplemental Dataset S23. List of Arabidopsis genes with a putative involvement in lipid metabolism.
Supplemental Dataset S24. Transcriptomics: list of all detected genes with putative lipid function.